Renal ischemia-reperfusion (IR) is a leading cause of acute kidney injury (IR-AKI), a significant unsolved clinical problem. A critical barrier to progress with IR-AKI is that there are gaps in our scientific understanding of the mechanisms underlying the persistent reduction of medullary blood flow (MBF), a key factor determining the outcome of IR-AKI and the progression to chronic kidney disease (CKD). Our goal is to address this critical barrier by focusing on the role of sphingosine-1-phosphate (S1P) as a potential signaling molecule contributing to renal microvascular dysfunction in IR-AKI. Our central hypothesis is that renal ischemia-reperfusion leads to significantly enhanced sensitivity of juxtamedullary afferent arterioles, the crucial vascular segment that controls MBF, to S1P-mediated vasoconstriction which contributes to a persistent reduction of MBF and a steady decline in glomerular filtration rate (GFR) in IR-AKI. Our objectives are to address this central hypothesis by (1) determining the role of S1P in controlling juxtamedullary afferent arteriolar function and renal hemodynamics in IR-AKI; (2) establishing the cellular mechanisms of S1P- dependent regulation of afferent arteriolar reactivity in IR-AKI; and (3) determining the pathophysiological role of S1P in the development of renal microvascular dysfunction in IR-AKI. We will test this central hypothesis through three specific aims. AIM 1 will test the hypothesis that renal ischemia-reperfusion leads to enhanced sensitivity of afferent arterioles to S1P which contributes to a persistent vasoconstriction and reduction of MBF in IR-AKI. AIM 2 will test the hypothesis that the renal ischemia-reperfusion-induced increase in reactive oxygen species production contributes to enhanced S1P sensitivity of afferent arterioles in IR-AKI. AIM 3 will test the hypothesis that inhibition of S1P receptor activation prevents enhancement of S1P-mediated afferent arteriolar vasoconstriction during ischemia-reperfusion and protects against IR-AKI. We will use the in vitro blood-perfused juxtamedullary nephron technique to assess the impact of IR on S1P-mediated arteriolar response in rats and in S1P2 receptor knockout mice. We will determine the influence of S1P on total and regional renal blood flow and GFR with IR-AKI. We will determine which S1P receptors contribute to enhanced S1P-mediated vasoconstriction. We will measure sphingolipid metabolites in kidney and plasma of IR rats or mice. Our outcomes will provide new mechanistic insights that renal IR activates the S1P signaling pathway in the renal microvasculature. Exogenous S1P causes potent vasoconstriction of afferent arterioles and reduction of renal blood flow and MBF, which will be enhanced in IR-AKI. S1P2 receptor blockade or deletion will protect against IR-AKI. The results of this study will improve our understanding of the pathophysiological mechanisms underlying the persistent reduction of MBF and a steady decline in GFR in IR-AKI. Improving MBF by inhibiting S1P2R signaling may represent a new therapeutic target for treating IR-AKI and preventing progression to CKD, thereby having a major impact in the field.